PROJECT SUMMARY While acute pain is an important biological signal in response to injured tissue, chronic pain occurs when the pain signaling outlasts the initial injury and has deleterious effects on health and quality of life. Chronic pain represents an enormous public health burden with few therapeutic options. Chronic pain is distinct from acute pain with several unique features including long-lasting activation of astrocytes. Astrocytes are CNS cells with diverse functions including energy homeostasis, regulation of the blood brain barrier, clearance of neurotransmitters, and regulation of synaptic transmission, all of which are altered in activated states. Preventing activation of astrocytes represents a key therapeutic target. Microglia, the resident immune cells of the CNS, have been implicated as key mediators of astrocyte activation. In this way, microglia manipulation may provide a tool to prevent or alter astrocyte activation, which in turn may prevent pain from becoming chronic. Previous research from our lab has shown that depletion of microglia at the time of transition from acute pain to chronic pain prevents chronic pain. However, when microglia are depleted once chronic pain is established, there are only transient improvements in pain-like behaviors. One explanation for these different effects is that microglia may be contributing indirectly to chronic pain by triggering astrocyte activation during the transition to chronic pain. However, once astrocytes are activated, microglia cease to have an active role in pain signaling and the alterations in spinal cord circuits maintaining chronic pain are due to changes in astrocyte function. I hypothesize that at the acute-to-chronic transition microglia are necessary and sufficient to activate astrocytes and that microglia effects in chronic pain are entirely dependent on astrocyte activation. In Aim 1, I will characterize astrocyte activation in a mouse model of pain-producing peripheral injury after selective depletion of microglia at the acute-to-chronic pain transition. I will further use DREADDs to exogenously activate astrocytes at the acute-to-chronic transition to determine if exogenous activation of astrocytes is sufficient to maintain the transition to chronic pain in the context of microglia depletion. In Aim 2, I will use exogenous activation of microglia in a naïve mouse to determine if activation of microglia is sufficient to activate astrocytes and if this activation of astrocytes leads to pain-like behaviors. Finally, in Aim 3 I will determine which signals from microglia are important for astrocyte activation in the induction of chronic pain using cell-cell interaction analyses of single nuclei RNA-Sequencing data from astrocytes and microglia. Using the innovative experiments in this research proposal, I will uncover the relative roles and contributions of microglia and astrocytes to chronic pain and generate new targets for pain therapeutics. The proposed research i...